Soluble immune checkpoint factors reflect exhaustion of antitumor immunity and response to PD-1 blockade

BACKGROUND Precise stratification of patients with non–small cell lung cancer (NSCLC) is needed for appropriate application of PD-1/PD-L1 blockade therapy. METHODS We measured soluble forms of the immune-checkpoint molecules PD-L1, PD-1, and CTLA-4 in plasma of patients with advanced NSCLC before PD-1/PD-L1 blockade. A prospective biomarker-finding trial (cohort A) included 50 previously treated patients who received nivolumab. A retrospective observational study was performed for patients treated with any PD-1/PD-L1 blockade therapy (cohorts B and C), cytotoxic chemotherapy (cohort D), or targeted therapy (cohort E). Plasma samples from all patients were assayed for soluble immune-checkpoint molecules with a highly sensitive chemiluminescence-based assay. RESULTS Nonresponsiveness to PD-1/PD-L1 blockade therapy was associated with higher concentrations of these soluble immune factors among patients with immune-reactive (hot) tumors. Such an association was not apparent for patients treated with cytotoxic chemotherapy or targeted therapy. Integrative analysis of tumor size, PD-L1 expression in tumor tissue (tPD-L1), and gene expression in tumor tissue and peripheral CD8+ T cells revealed that high concentrations of the 3 soluble immune factors were associated with hyper or terminal exhaustion of antitumor immunity. The combination of soluble PD-L1 (sPD-L1) and sCTLA-4 efficiently discriminated responsiveness to PD-1/PD-L1 blockade among patients with immune-reactive tumors. CONCLUSION Combinations of soluble immune factors might be able to identify patients unlikely to respond to PD-1/PD-L1 blockade as a result of terminal exhaustion of antitumor immunity. Our data suggest that such a combination better predicts, along with tPD-L1, for the response of patients with NSCLC. TRIAL REGISTRATION UMIN000019674. FUNDING This study was funded by Ono Pharmaceutical Co. Ltd. and Sysmex Corporation.


Study design and patients
The overall design of the study is outlined in Figure 1.From December 2015 to September 2016, 50 previously treated patients with advanced or recurrent NSCLC were prospectively enrolled in a phase 2 biomarker-finding trial, Nivolution, that was conducted at Kindai University Hospital.Patients were eligible for enrollment if an archival tumor tissue specimen obtained within 1 year before enrollment or newly biopsied tissue was available.Nivolumab (3 mg/kg) was administered intravenously biweekly.Radiologic imaging was performed every 6 weeks.Tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (1).The study protocol was approved by the ethics committee at Kindai University Hospital and Kyoto University Hospital.Each patient provided written informed consent before enrollment.
For the cohorts B and C, patients with advanced or recurrent NSCLC receiving antibodies to PD-1 or to PD-L1-including nivolumab, pembrolizumab, and atezolizumab-were enrolled for a retrospective study conducted at Kindai University Hospital, Kyoto University Hospital and Izumi City General Hospital.Also, for the cohort D and E, patients with advanced or recurrent NSCLC receiving cytotoxic chemotherapy without ICB therapy or TKIs as an initial therapy, respectively, were retrospectively enrolled at Kindai University Hospital and Kyoto University Hospital.
Blood samples and medical records were obtained for all patients.Tumor response was assessed by computed tomography every 6 to 12 weeks according to RECIST version 1.1.These studies were conducted according to the Declaration of Helsinki, and the protocols were approved by the Institutional Review Board of each hospital.

Immunohistochamistry
Tumor histology was classified according to WHO criteria (2).In the Nivolution trial, sections of formalin-fixed paraffin-embedded tumor tissue were subjected to IHC with monoclonal antibodies to PD-L1 (kit with clone 28-8, Abcam) and to CD8 (clone C8/144B, Dako).The percentage of tumor cells positive for PD-L1 (tPD-L1) was determined as previously described (3,4).TILs were evaluated on the basis of staining for CD8.Tumor tissue samples including at least 100 viable tumor cells were eligible for assessment of TILs.The number of TILs was determined at an absolute magnification of 400× (0.20 mm 2 per field).At least one and a maximum of five scanned fields of tumor regions were randomly chosen for each TIL count.TILs were counted by a board-certified pathologist, and the density of TILs in each tumor was calculated by dividing the number of TILs by the viewed fields (4).The cutoff value of 12.0/field was determined on the basis of the median number of tumor-infiltrated CD8 + T cells per field.

Gene expression analysis by RNA-seq
The RNA extracted from tissue samples and blood cells was subjected to reverse transcription with the use of a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific), and the resulting cDNA was subjected to multiplex PCR amplification, end repair, and ligation of barcoded adaptors.Pooled libraries were processed with an Ion Chef System (Thermo Fisher Scientific) for template preparation.Libraries were then loaded onto an Ion 550 chip and sequenced with the Ion S5 XL sequencing system.Ion Torrent Suite v5.10 software (Thermo Fisher Scientific) was used for base calling, alignment to the human reference genome (hg19), and quality control.Raw reads were analyzed automatically with the AmpliSeqRNA plugin to generate gene-level expression values for all 20,802 RefSeq human genes.
Acquisition of samples was performed with a BD FACSCanto II cell analyzer (BD Biosciences).Data were collected with the use of BD FACSDiva software version 6.1.3and further analyzed with FlowJo 10.4 (Tree Star).

Microarray analysis of peripheral CD8 + T cells and gene enrichment analysis
CD8 + T cells were purified from PBMCs with an AutoMACS system (Miltenyi Biotec).
Total RNA was isolated from the cells with an RNeasy Micro Prep Kit (Qiagen), and its quality was analyzed with TapeStation (Agilent).Portions (5 ng) of the total RNA were labeled with the use of a GeneChip WT Pico Reagent Kit (Thermo Fisher Scientific) and subjected to hybridization with a Human GeneChip Clariom D Array (Thermo Fisher Scientific).The array data were analyzed with Signal Space Transformation-Robust Multichip Analysis (SST-RMA) and Sketch-Quantile normalization (Expression Console Software).

Cytokine analysis
Plasma samples were obtained by centrifugation of EDTA-treated whole blood at 2400 × g for 10 min at 4°C.Concentrations of the cytokines shown in Figure 6D were measured with V-PLEX Plus Proinflammatory Panel 1, Cytokine Panel 1, V-PLEX Plus Cytokine Panel 1 (Human), V-PLEX Plus Chemokine Panel 1 (Human), and Human ELISA Kits (Meso Scale Discovery Electrochemiluminescence Service).All assays were performed in triplicate.A correlation matrix for the plasma concentrations of the cytokines as well as those of sPD-1, sPD-L1, and sCTLA-4 was generated by Ward's clustering with squared Euclidean distances.

Determination of the cutoff values defining high versus low concentrations of each soluble factor
The cutoff values for soluble factor concentrations were determined with a proportional hazards model.A Cox proportional hazards model was thus fitted to the PFS data in order to estimate the HR for each covariate of interest.After sorting according to the biomarker values, dummy variables such as those shown in Supplemental Methods Table 1 below were generated.

Table 2
From the latter table, DB(X) for which the absolute value of log[HR]is maximum was identified.The point in Supplemental Methods Table1corresponding to the identified DB(X) is the cutoff point.For example, if DB(4) gives the maximum absolute value of log[HR], the cutoff point is the value between 25 and 26 in Supplemental Methods